Long-chain fatty acyl coenzyme A inhibits NME1/2 and regulates cancer metastasis

Significance The study provided a long-sought molecular mechanism that could explain the link between fatty acid metabolism and cancer metastasis. Further understanding may lead to new strategies to inhibit cancer metastasis. The chemical proteomic approach developed here will be useful for discovering other regulatory mechanisms of protein function by small molecule metabolites.

2 dissolved in CH3CN (1 mL) and cooled down to 0 °C. (Trimethylsilyl)diazomethane (2.0 M Et2O solution) (0.3 mL, 1.5 eq.) was added slowly and the mixture was allowed to gradually warm up to room temperature and vigorously stirred for 16 h under N2.
After the reaction was completed (monitored by TLC), the solvent was removed under reduced pressure and the residue was dissolved in Et2O (15 mL). Hydrogen bromide (37% in H2O, 10.0 eq.) was added slowly and the mixture was refluxed for 3 h. Saturated NaHCO3 solution (15 mL) was added to quench the reaction. The organic phase was separated from the aqueous phase, and the aqueous phase was extracted with EtOAc (20 mL × 2). The combined organic phase was dried over Na2SO4. After concentration in vacuo, the residue was subsequently purified through column chromatography (hexanes/Et2O: from 15:1 to 9:1) to afford S1 as a colorless semi-solid (90 mg, 69% yield from Alk14).
To a 25 mL round bottom flask equipped with a stir bar was added coenzyme A trilithium salt (45 mg, 0.056 mmol, 1.0 eq.) and DL-dithiothreitol (1.0 mg, 0.0065 mmol, 0.11 eq.). After the flask was evacuated and backfilled with N2 twice, Na2CO3 aqueous solution (0.04 M, 5 mL) were added via a syringe. The mixture was stirred for 30 min at room temperature and then a solution of S1 (56 mg, 0.17 mmol, 3.0 eq.) in tert-butanol (7 mL) was added. The reaction mixture was stirred for 16 h under room temperature. The solvent was removed under reduced pressure and the residue was 3 dissolved in water (15 mL). The mixture was filtered through a pad of Celite and the filtrate was frozen and lyophilized to remove water. The obtained crude white solid was directly used in the next step without further purification.
To a 2-dram vial equipped with a stir bar was added the crude S2 (5.6 μmol, 1.0 eq.).
After the vial was evacuated and backfilled with N2 twice, degassed water (1.0 mL) and MeOH (1.0 mL) were added via a syringe. Subsequently, NaHCO3 (100 μL, 8.4 mM stock solution), CuSO4•5H2O (100 μL, 0.56 mM stock solution), sodium ascorbate (100 μL, 1.12 mM stock solution) and azide-PEG3-biotin conjugate (170 μL, 50 mM stock solution) were added and the reaction mixture was stirred at room temperature for 12 h and monitored by LC-MS. After concentration in vacuo, the residue was redissolved in H2O   L-arginine, and 10% dialyzed FBS for 5 generations. The"heavy" and "light" cells were collected and lysed in 1% NP40 lysis buffer separately, according to the workflow described above. Protein input of 8 mg for each "heavy" and "light" total lysate was treated with either DMSO or 25 M of C14-CoA-biotin probe at 4 o C for 1h. Then both "heavy" and "light" samples were subjected for streptavidin immunoprecipitation separately. After washing the affinity gel three times with NP40 washing buffer, "heavy" and "light" samples were mixed and washed two more times with NP40 washing buffer.
Then on-beads trypsin digestion was conducted and the digested peptide solutions were NDPK activity assay. NDPK assay was performed in the kinase buffer (50mM triethanolamine, 150mM NaCl, 2mM MgCl2, 1mM EDTA and 1mM DTT) with 50nM of purified NME proteins, 500uM GDP and 1mM ATP at room temperature for 5 min. Diffraction images were collected at the Advanced Photon Source, NE-CAT beamline.
The data was processed by XDS 5 and Aimless 6 , and the structure was solved by molecular replacement with Phaser 7 as part of the NECAT beamline's RAPD pipeline.
PDB entry 3BBF was used as the model. 8             Transferrin internalization was monitored and quantified by Cytation 5 imaging station.

Supplementary Figures and Legends
Statistical significance was calculated by unpaired two-tailed Student's t test.